The collection and storage of biological fluids, such as blood, is well represented by neonatal testing of infants for phenylketourionic acid (PKU). The heel of a newborn child is pricked by a lance. A piece of cellulose paper is applied to the blood spot. The spot is allowed to dry before being sent to a laboratory for testing. Almost all of the neonatal testing for PKU is performed in this manner. However, such a technique is not easily applicable to the collection and storage of biologically related fluids when one desires to analyze any nucleic acids present in the biological source. One would have to use a protease digestion, organic extraction, and/or an ion exchange step in order to retrieve nucleic acids.
Nucleic acids, such as deoxyribonucleic acids (DNA) or ribonucleic acids (RNA), have become of increasing interest as analytes for clinical or forensic uses. Powerful new molecular biology technologies enable one to detect for congenital diseases or infectious diseases. These same technologies can characterize DNA for use in settling factual issues in legal proceedings such as paternity suits and criminal prosecutions. Nucleic acid testing has been made possible due to powerful amplification methods. One can take small amounts of nucleic acids which, in and of themselves would be undetectable, and increase or amplify the amount to a degree where useful amounts are present for detection.
The most commonly employed amplification technique is known as polymerase chain reaction, (PCR). Nucleic acid polymerases are used with template DNA from the sample in a cycled manner to create greater amounts of a starting nucleic acid materials, which are easily detected. One of ordinary skill in the art knows that the effectiveness and reproducibility of PCR amplification is dependent, in part, on the purity and amount of the DNA template. Certain molecules present in biological sources of nucleic acids are known to stop or inhibit PCR amplification. For example, in whole blood, hemoglobin is known to inhibit PCR reactions. Thus, the removal or inactivation of such inhibitors is a key factor in performing PCR reactions.
A method for storing DNA is disclosed in U.S. Pat. No. 5,496,562 to Leigh A. Burgoyne. An absorbent cellulose based matrix is treated with a combination of a weak base, a chelating agent, an anionic detergent, and, optionally, uric acid. The resulting product has an alkaline pH. DNA binds to this matrix and is protected against degradation.
A process for isolating nucleic acids is shown in U.S. Pat. No. 5,234,809 to William R. Boom et alia, (Boom). Recognizing that typical biological sources of nucleic acids can affect PCR reactions, Boom discloses using a combination of a biological source material, chaotropic salt, and a solid support, preferably finely divided glass. All three elements are combined in a liquid mixing device, with any nucleid acids present binding to the glass. After mixing, the solid support must be removed from the mixing device, washed, and the template nucleic acid eluted. Only then can it be exposed to amplification reactions.
Chaotropic salts have been used in association with isolating RNA. U.S. Pat. No. 4,483,920 to David Gillespie et alia, discloses a method for immobilizing messenger RNA onto filter material. Cellular components are solubilized using a chaotropic salt. The solubilized components are then passed through a filter, the messenger RNA selectively binding to the filter. The filter and RNA are baked prior to measurement by a labeled probe. Another method is shown by David Gillespie et alia, in U.S. Pat. No. 5,155,018. Here, RNA-containing sources are contacted with finely-divided glass in the presence of a binding solution comprising concentrated, acidified chaotropic salts. Under these conditions, RNA, but not DNA, binds selectively to the glass.
A poster disclosure at the annual American Association of Clinical Chemistry in 1995 by Dr. Michael A. Harvey et alia revealed that chaotropic salts can be used to prepare DNA from dried and untreated whole blood spots for PCR amplification. Hemoglobin present in dried untreated whole blood spots was known to cause an inhibition of PCR reactions. A cellulosic paper treated with a chaotropic salt was found to overcome the problem of hemoglobin inhibition in untreated whole blood spots.